US6451980B1 - Signal enhancement of bispecific antibody-polymer probe for immunoassay use - Google Patents

Signal enhancement of bispecific antibody-polymer probe for immunoassay use Download PDF

Info

Publication number
US6451980B1
US6451980B1 US09/380,168 US38016899A US6451980B1 US 6451980 B1 US6451980 B1 US 6451980B1 US 38016899 A US38016899 A US 38016899A US 6451980 B1 US6451980 B1 US 6451980B1
Authority
US
United States
Prior art keywords
antibody
bispecific antibody
probe
bispecific
polymer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
US09/380,168
Other versions
US20020031781A1 (en
Inventor
Ban-an Khaw
Jagat Narula
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KHAW DR BAN-AN
Akrivis Technologies LLC
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US09/380,168 priority Critical patent/US6451980B1/en
Assigned to KHAW, BAN-AN reassignment KHAW, BAN-AN ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NARULA, JAGAT
Priority to US09/727,421 priority patent/US20010024795A1/en
Assigned to BIOSPECIFIC LLC reassignment BIOSPECIFIC LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KHAW, DR. BAN-AN,
Priority to US10/071,397 priority patent/US20020119582A1/en
Publication of US20020031781A1 publication Critical patent/US20020031781A1/en
Publication of US6451980B1 publication Critical patent/US6451980B1/en
Application granted granted Critical
Assigned to KHAW, DR. BAN-AN reassignment KHAW, DR. BAN-AN ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BIOSPECIFIC LLC
Assigned to AKRIVIS TECHNOLOGIES, LLC reassignment AKRIVIS TECHNOLOGIES, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KHAW, BAN-AN
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/817—Enzyme or microbe electrode
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/912—Absidia

Definitions

  • An immunoassay utilizes antibodies to detect a compound of choice.
  • the sensitivity of this detection is generally limited to the amount of signal that can be carried either on an antibody, for a direct binding assay, or on the probe compound, in a competitive inhibition assay.
  • existing immunoassays such as radioimmunoassays, ELISA, immunofluorescent assays or immunochemiluminescent assays
  • too many signal entities such as radioisotopes, horse radish peroxidase or alkaline phosphatase, attached to the detection moieties invariably inactivate the antibody or denature the antigen and change the property of he detection probe. Therefore, in order to obtain more signal, additional antibody or probe must be added. This, in turn, reduces the sensitivity of the assay, the capability of the assay to detect minute quantities of the compound in question.
  • the invention is directed to a method to increase the sensitivity of an immunoassay, by at least 10,000 fold, without losing specificity. This improvement is achieved by the use of a bispecific antibody complex and a unique detection signal probe capable of recognizing the bispecific antibody complex.
  • the invention features an immunoassay method including reacting a sample from a patient with a bispecific antibody, wherein the bispecific antibody includes one antibody specific for a compound to be detected and a second antibody specific for a compound foreign to said patient sample, i.e., non-endogenous, and subsequently reacting the patient sample with a polymer probe, wherein the polymer probe includes a compound recognized by the second antibody in the bispecific antibody complex and further includes at least two detectable signals.
  • the invention also features the bispecific antibody and the polymer probe of the method of the invention.
  • the sample from the patient is a blood or serum sample;
  • the bispecific antibody includes an antimyosin antibody and an antibody against DTPA;
  • the polymer probe is a polylysine polymer and includes DTPA and at least six HRP as the detectable signal compounds.
  • FIG. 1 a shows a standard ELISA according to the prior art
  • FIG. 1 b shows an immunoassay according to the invention.
  • FIG. 2 is a graph showing competitive inhibition curves using standard ELISA (R11D10), bispecific antibody complex with standard secondary antibody for signal production (BiMAb (Ab-HRP)), and the method according to the invention (BiMAb(PL-DTPA-HRP)).
  • the invention is directed to the development of a new approach to the use of bispecific antibodies in immunoassays.
  • the new specific antibody comprises one antibody specific for the compound associated with the pathological state to be detected and another antibody to a chemical or reporter compound that is not found naturally in man. These two are chemically or genetically linked.
  • the bispecific antibody complex constitutes the first line of interaction with the compound one is attempting to detect. Normally many antibodies must react with the compound to enable development of sufficient signal intensity for detection.
  • a novel detection probe is used, made up of any type polymer, such as polylysine or other polyamino acid, that is amenable to attachment of signal reagents and reporter compounds.
  • the amount of signal reagent that can be used in a given assay is limited only by the size of the polymer. only a few molecules of the detection probe are therefore needed to provide this signal.
  • the signal probe is extremely versatile as any type of signal producing compound such as radioactivity, chemical color producing enzymes or fluorescent probes can be attached to the polymer backbone. Signal amplification is not limited by the nature of the bispecific antibody complex itself.
  • the immunoassay sensitivity can be amplified by at least 10,000-fold compared to conventional immunoassays or immunosandwich assays. Since early detection of many pathological states, such as acute myocardial infarction and cancer, is limited by the sensitivity of immunoassays to detect minute elevations of the pathologically associated compounds, an method and compounds of the invention will enable diagnosis of disease states at a much earlier time than previous assays, which may allow for better therapeutic intervention.
  • Another advantage of the method of the invention is the versatility for adaptation to any antibody.
  • the method could be adapted to detect troponin-I or T by using the antibody specific for troponin-I or T attached to a second antibody, such as the antibodies shown herein, that recognizes the detector probe. If higher sensitivity is necessary, the polymer probe could be generated to carry higher numbers of signal compounds.
  • the polymer probe can include any kind of signal compound, such as radioisotope, fluorescent, or paramagnetic linked signal compounds.
  • Serum immunoassays for intracardiac contractile proteins constitute the mainstay for detection of myocyte necrosis associated with various cardio-vascular disorders.
  • myosin heavy chain (MHC) fragments can be detected by immunoassay only after 48 h from the onset of chest pain.
  • MAb monoclonal antibody
  • MAb 4G4-1D5 specific for DTPA.
  • the probe consisted of DTPA-modified polylysine (28:1 molar ratio) covalently linked to horse-radish peroxidase (6 moles/mole polylysine) (PL-DTPA-HRP).
  • Porcine cardiac myosin (PCM, 1 ⁇ /ml) was used to coat the microtiter wells. After overnight incubation and washing, three times, 50 ⁇ l each of 5 ⁇ g/ml BiMAbor MAb and serial dilutions of PCM (0.001 to 100 ⁇ g/ml) or 50 ⁇ l of serial dilutions (1/1 to 1/10000) of patient sera pre-incubated for 1 h at 37° C. were added and incubated for 2 h at 37° C. After washing, the wells were incubated with goat-antimouse IgG-HRP or PL-DTPA-HRP for 2 h. A chromogen, dinitrobenzidine was used to develop the assay.
  • PCM Porcine cardiac myosin
  • BiMAb and R11D10 were the same at 1.5 ⁇ 10 9 L/mole.
  • the sensitivity of BiMAb was 0.5 ng, whereas that of R11D10 was 0.5 ⁇ g (1 ⁇ g/ml).
  • BiMAb developed with the conventional goat anti-mouse IgG-HRP had a sensitivity of 0.05 ⁇ g. Therefore, BiMAb assay has a 1000 fold increase in sensitivity compared to the conventional immunoassay in the sera of 3 heart transplant patients. Using the BiMAb assay, 2.5, 1.25 and 1.3 ng MHC/50 ⁇ l serum at ⁇ fraction (1/10) ⁇ 3 dilution, were detected.
  • This BiMAb technology can be used in RIA or ELISA by interchanging the HRP probe for radiolabeled probe and should provide more specific in vitro diagnosis of acute myocardial infarction since detection of MHC is not feasible at the present time of day 1 of myocardial infarction by conventional immunoassays.
  • the DTPA-modified polylysine probe of Example I was covalently linked to 12 moles of horse-radish peroxidase per mole of polylysine.
  • the results of the study show that the sensitivity of the bispecific assay of the invention (10 ⁇ 5 to 100 ⁇ g/ml) was at least 10,000 fold better than the conventional immunoassay (0.1 ⁇ g/ml).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

An immunoassay method including reacting a sample from a patient with a bispecific antibody, wherein the bispecific antibody includes one antibody specific for a compound to be detected and a second antibody specific for a compound foreign to said patient sample, and subsequently reacting the patient sample with a polymer probe, wherein the polymer probe includes a compound recognized by the second antibody in the bispecific antibody complex and further includes at least two detectable signals; the bispecific antibody; and the polymer probe of the immunoassay method are disclosed.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 60/039,111, filed Feb. 26, 1997, and to International Application No. PCT/US98/03638, filed Feb. 25, 1998. Each of the applications cross-referenced in this section are incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT BACKGROUND OF THE INVENTION
An immunoassay utilizes antibodies to detect a compound of choice. However, the sensitivity of this detection is generally limited to the amount of signal that can be carried either on an antibody, for a direct binding assay, or on the probe compound, in a competitive inhibition assay. For example, in existing immunoassays, such as radioimmunoassays, ELISA, immunofluorescent assays or immunochemiluminescent assays, too many signal entities, such as radioisotopes, horse radish peroxidase or alkaline phosphatase, attached to the detection moieties invariably inactivate the antibody or denature the antigen and change the property of he detection probe. Therefore, in order to obtain more signal, additional antibody or probe must be added. This, in turn, reduces the sensitivity of the assay, the capability of the assay to detect minute quantities of the compound in question.
For all existing immunoassays, there is lag time for the compound of interest to reach a high enough concentration in the serum to become detectable for diagnostic purposes. In the case of heart attacks, there is a delay of 4-6 hours from the onset of chest pain until the diagnostic detection of CK-MB, Troponin-T or I is possible. Myoglobin is detectable earlier, but its specificity is low. If there were an assay that could detect very minute increases of these indicator compounds in the blood at an earlier point in time, then therapeutic intervention could be started earlier and thereby bring about greater myocardial salvage. In the case of cancer detection, where, e.g., tumor associated antigens related to breast cancer or colon cancer, etc., are detected, treatment might be more effective if minute elevations of these antigens could be detected at an early stage. Therefore, there is a need to increase the sensitivity of the assay without adversely affecting the specificity of the assay system.
SUMMARY OF THE INVENTION
The invention is directed to a method to increase the sensitivity of an immunoassay, by at least 10,000 fold, without losing specificity. This improvement is achieved by the use of a bispecific antibody complex and a unique detection signal probe capable of recognizing the bispecific antibody complex.
In one aspect, the invention features an immunoassay method including reacting a sample from a patient with a bispecific antibody, wherein the bispecific antibody includes one antibody specific for a compound to be detected and a second antibody specific for a compound foreign to said patient sample, i.e., non-endogenous, and subsequently reacting the patient sample with a polymer probe, wherein the polymer probe includes a compound recognized by the second antibody in the bispecific antibody complex and further includes at least two detectable signals. The invention also features the bispecific antibody and the polymer probe of the method of the invention. Preferably, the sample from the patient is a blood or serum sample; the bispecific antibody includes an antimyosin antibody and an antibody against DTPA; and the polymer probe is a polylysine polymer and includes DTPA and at least six HRP as the detectable signal compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims, taken in conjunction with the accompanying drawings, in which:
FIG. 1a shows a standard ELISA according to the prior art;
FIG. 1b shows an immunoassay according to the invention; and
FIG. 2 is a graph showing competitive inhibition curves using standard ELISA (R11D10), bispecific antibody complex with standard secondary antibody for signal production (BiMAb (Ab-HRP)), and the method according to the invention (BiMAb(PL-DTPA-HRP)).
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to the development of a new approach to the use of bispecific antibodies in immunoassays. The new specific antibody comprises one antibody specific for the compound associated with the pathological state to be detected and another antibody to a chemical or reporter compound that is not found naturally in man. These two are chemically or genetically linked. The bispecific antibody complex constitutes the first line of interaction with the compound one is attempting to detect. Normally many antibodies must react with the compound to enable development of sufficient signal intensity for detection. However, in the method of the invention, a novel detection probe is used, made up of any type polymer, such as polylysine or other polyamino acid, that is amenable to attachment of signal reagents and reporter compounds. The amount of signal reagent that can be used in a given assay is limited only by the size of the polymer. only a few molecules of the detection probe are therefore needed to provide this signal. The signal probe is extremely versatile as any type of signal producing compound such as radioactivity, chemical color producing enzymes or fluorescent probes can be attached to the polymer backbone. Signal amplification is not limited by the nature of the bispecific antibody complex itself.
Therefore, the immunoassay sensitivity can be amplified by at least 10,000-fold compared to conventional immunoassays or immunosandwich assays. Since early detection of many pathological states, such as acute myocardial infarction and cancer, is limited by the sensitivity of immunoassays to detect minute elevations of the pathologically associated compounds, an method and compounds of the invention will enable diagnosis of disease states at a much earlier time than previous assays, which may allow for better therapeutic intervention.
Another advantage of the method of the invention is the versatility for adaptation to any antibody. For example, the method could be adapted to detect troponin-I or T by using the antibody specific for troponin-I or T attached to a second antibody, such as the antibodies shown herein, that recognizes the detector probe. If higher sensitivity is necessary, the polymer probe could be generated to carry higher numbers of signal compounds. Furthermore, the polymer probe can include any kind of signal compound, such as radioisotope, fluorescent, or paramagnetic linked signal compounds.
All previously existing ELISA radioimmunoassays, dipstick assays for cancer, pregnancy, serum enzymes and probes and any assays utilizing antibodies could be modified according to the method of the invention to provide enhanced sensitivity. In addition, in vivo application to enhance target signal by using the method of the invention is also possible.
The following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same. These examples are not intended in any way otherwise to limit the scope of the disclosure.
EXAMPLE I
Serum immunoassays for intracardiac contractile proteins constitute the mainstay for detection of myocyte necrosis associated with various cardio-vascular disorders. However, myosin heavy chain (MHC) fragments can be detected by immunoassay only after 48 h from the onset of chest pain. To enhance immunodetection of MHC, monoclonal antibody (MAb) R11D10 specific for cardiac MHC was covalently linked to MAb 4G4-1D5 specific for DTPA. The probe consisted of DTPA-modified polylysine (28:1 molar ratio) covalently linked to horse-radish peroxidase (6 moles/mole polylysine) (PL-DTPA-HRP). Porcine cardiac myosin (PCM, 1 μ/ml) was used to coat the microtiter wells. After overnight incubation and washing, three times, 50 μl each of 5 μg/ml BiMAbor MAb and serial dilutions of PCM (0.001 to 100 μg/ml) or 50μl of serial dilutions (1/1 to 1/10000) of patient sera pre-incubated for 1 h at 37° C. were added and incubated for 2 h at 37° C. After washing, the wells were incubated with goat-antimouse IgG-HRP or PL-DTPA-HRP for 2 h. A chromogen, dinitrobenzidine was used to develop the assay. The affinity of BiMAb and R11D10 were the same at 1.5×109 L/mole. The sensitivity of BiMAb was 0.5 ng, whereas that of R11D10 was 0.5 μg (1 μg/ml). BiMAb developed with the conventional goat anti-mouse IgG-HRP had a sensitivity of 0.05 μg. Therefore, BiMAb assay has a 1000 fold increase in sensitivity compared to the conventional immunoassay in the sera of 3 heart transplant patients. Using the BiMAb assay, 2.5, 1.25 and 1.3 ng MHC/50 μl serum at {fraction (1/10)}3 dilution, were detected. This BiMAb technology can be used in RIA or ELISA by interchanging the HRP probe for radiolabeled probe and should provide more specific in vitro diagnosis of acute myocardial infarction since detection of MHC is not feasible at the present time of day 1 of myocardial infarction by conventional immunoassays.
EXAMPLE II
In a subsequent experiment the DTPA-modified polylysine probe of Example I was covalently linked to 12 moles of horse-radish peroxidase per mole of polylysine. The results of the study show that the sensitivity of the bispecific assay of the invention (10−5 to 100 μg/ml) was at least 10,000 fold better than the conventional immunoassay (0.1 μg/ml).
While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.

Claims (6)

What is claimed is:
1. A bispecific antibody complex comprising:
a bispecific antibody comprising a first antibody specific for a first antigen to be assayed in a sample, said first antibody being linked to a second antibody specific for a second antigen that is not endogenous to the species where said sample originates, and said bispecific antibody being non-covalently bound to a polymer probe, said probe comprising each of the following: a polymer backbone; a second antigen which is linked to said polymer backbone and which is recognizable by said second antibody; at least two detectable signal compounds linked to said polymer backbone.
2. A bispecific antibody complex according to claim 1 wherein said first and second antibodies are covalently linked in said bispecific antibody.
3. A bispecific antibody complex according to claim 1, wherein said polymer backbone is covalently linked to both said second antigen and said signal compounds.
4. The bispecific complex of claim 1 wherein said first and second antibodies each consist of one heavy and one light chain.
5. The bispecific complex of claim 1 further comprising a polymer probe being non-covalently bound to said bispecific antibody, said probe comprising each of the following: a polymer backbone; the non-endogenous second antigen which is linked to said polymer backbone and which is recognizable by said second antibody; at least two detectable signal compounds linked to said polymer backbone.
6. The bispecific complex of claim 4 further comprising a polymer probe being non-covalently bound to said bispecific antibody, said probe comprising each of the following: a polymer backbone; the non-endogenous second antigen which is linked to said polymer backbone and which is recognizable by said second antibody; at least two detectable signal compounds linked to said polymer backbone.
US09/380,168 1997-02-26 1998-02-25 Signal enhancement of bispecific antibody-polymer probe for immunoassay use Expired - Lifetime US6451980B1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US09/380,168 US6451980B1 (en) 1997-02-26 1998-02-25 Signal enhancement of bispecific antibody-polymer probe for immunoassay use
US09/727,421 US20010024795A1 (en) 1997-02-26 2000-12-01 Immunoassay technique using multispecific molecules
US10/071,397 US20020119582A1 (en) 1997-02-26 2002-02-06 Signal enhancement of bispecific antibody-polymer probe for immunoassay use

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US3911197P 1997-02-26 1997-02-26
PCT/US1998/003638 WO1998038513A1 (en) 1997-02-26 1998-02-25 Signal enhancement of bispecific antibody-polymer probe for immunoassay use
US09/380,168 US6451980B1 (en) 1997-02-26 1998-02-25 Signal enhancement of bispecific antibody-polymer probe for immunoassay use

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/727,421 Continuation-In-Part US20010024795A1 (en) 1997-02-26 2000-12-01 Immunoassay technique using multispecific molecules
US10/071,397 Division US20020119582A1 (en) 1997-02-26 2002-02-06 Signal enhancement of bispecific antibody-polymer probe for immunoassay use

Publications (2)

Publication Number Publication Date
US20020031781A1 US20020031781A1 (en) 2002-03-14
US6451980B1 true US6451980B1 (en) 2002-09-17

Family

ID=21903743

Family Applications (2)

Application Number Title Priority Date Filing Date
US09/380,168 Expired - Lifetime US6451980B1 (en) 1997-02-26 1998-02-25 Signal enhancement of bispecific antibody-polymer probe for immunoassay use
US10/071,397 Abandoned US20020119582A1 (en) 1997-02-26 2002-02-06 Signal enhancement of bispecific antibody-polymer probe for immunoassay use

Family Applications After (1)

Application Number Title Priority Date Filing Date
US10/071,397 Abandoned US20020119582A1 (en) 1997-02-26 2002-02-06 Signal enhancement of bispecific antibody-polymer probe for immunoassay use

Country Status (3)

Country Link
US (2) US6451980B1 (en)
EP (1) EP0981748A4 (en)
WO (1) WO1998038513A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040071696A1 (en) * 2002-04-05 2004-04-15 The Regents Of The University Of California Bispecific single chain Fv antibody molecules and methods of use thereof
US20050100543A1 (en) * 2003-07-01 2005-05-12 Immunomedics, Inc. Multivalent carriers of bi-specific antibodies
US20060099205A1 (en) * 2002-04-05 2006-05-11 The Regents Of The University Of California Bispecific single chain FV antibody molecules and methods of use thereof
US20100196265A1 (en) * 2006-11-21 2010-08-05 Adams Gregory P Anti-egfr family antibodies, bispecific anti-egfr family antibodies and methods of use thereof
US8003373B2 (en) 2003-04-25 2011-08-23 Medtronic, Inc. Optical detector for enzyme activation

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6753189B1 (en) * 1998-06-04 2004-06-22 Mizuho Medy Co., Ltd. Detection apparatus and method for the same
GB9819411D0 (en) * 1998-09-04 1998-10-28 Ks Biomedix Ltd Antibodies
DK1340086T3 (en) * 2000-10-17 2008-12-01 Besst Test Aps Assay for direct detection of an RS virus-related biological cell in a body fluid sample
WO2005019820A1 (en) * 2003-08-25 2005-03-03 Marc Ramael A method and kit for the quantitative and/or qualitative detection of components in a sample
GB0320459D0 (en) * 2003-09-01 2003-10-01 Selective Antibodies Ltd Assay methods and materials
WO2009018576A1 (en) * 2007-08-02 2009-02-05 Biodesic Compositions and methods for analyte detection and quantitation
SG177560A1 (en) * 2009-07-06 2012-03-29 Hoffmann La Roche Bi-specific digoxigenin binding antibodies
US11340218B2 (en) 2016-05-25 2022-05-24 Kromnigon Ab Method for preparing a biological sample for use in an immunolabeling process

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990015993A1 (en) * 1989-06-14 1990-12-27 The General Hospital Corporation Assay for human ventricular myosin lc1 and monoclonal antibody thereto
US5223242A (en) 1985-11-05 1993-06-29 The General Hospital Corporation Negatively charged specific affinity reagents
WO1994012196A1 (en) * 1992-11-25 1994-06-09 Tanox Biosystems, Inc. Conjugates and constructs including anti-cd28 and anti-cd3 binding molecules
US5332567A (en) * 1989-08-24 1994-07-26 Immunomedics Detection and treatment of infections with immunoconjugates
US5482698A (en) * 1993-04-22 1996-01-09 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin polymer conjugates
US5591828A (en) * 1989-06-22 1997-01-07 Behringwerke Aktiengesellschaft Bispecific and oligospecific mono-and oligovalent receptors, the preparation and use thereof
US5698178A (en) * 1994-08-05 1997-12-16 Immunomedics, Inc. Polyspecific immunoconjugates and antibody composites for targeting the multidrug resistant phenotype
US5851527A (en) * 1988-04-18 1998-12-22 Immunomedics, Inc. Method for antibody targeting of therapeutic agents

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1929292A (en) * 1991-05-14 1992-12-30 Hybritech Incorporated Polymeric compositions having bound antibodies

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5223242A (en) 1985-11-05 1993-06-29 The General Hospital Corporation Negatively charged specific affinity reagents
US5851527A (en) * 1988-04-18 1998-12-22 Immunomedics, Inc. Method for antibody targeting of therapeutic agents
WO1990015993A1 (en) * 1989-06-14 1990-12-27 The General Hospital Corporation Assay for human ventricular myosin lc1 and monoclonal antibody thereto
US5591828A (en) * 1989-06-22 1997-01-07 Behringwerke Aktiengesellschaft Bispecific and oligospecific mono-and oligovalent receptors, the preparation and use thereof
US5332567A (en) * 1989-08-24 1994-07-26 Immunomedics Detection and treatment of infections with immunoconjugates
WO1994012196A1 (en) * 1992-11-25 1994-06-09 Tanox Biosystems, Inc. Conjugates and constructs including anti-cd28 and anti-cd3 binding molecules
US5482698A (en) * 1993-04-22 1996-01-09 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin polymer conjugates
US5698178A (en) * 1994-08-05 1997-12-16 Immunomedics, Inc. Polyspecific immunoconjugates and antibody composites for targeting the multidrug resistant phenotype

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Devys et al., "Comparative targeting of human colon-carcinoma multi-cell spheroids using one-and two-step (bispecific antibody) techniques", Int. J. Cancer, 67 883-891 (1996). These references were transmitted by the International Bureau.
Kranenborg et al., "Development and Characterization of anti-renal cell carcinoma X antichelate bispecific monoclonal antibodies for two-phase targeting of renal cell carcinoma", Canc. Res. 55, 23 Supplement 5864s-5867s (1995). These references were transmitted by the International Bureau.
Rosebrough, S.F., "Two step immunological approaches for imaging and therapy", Q.J. Nucl. Med., 40 234-251 (1996). These references were transmitted by the International Bureau.
Torchilin et al., "The antibody linked chelating polymers for nuclear therapy and diagnostics", Crit. Rev. Therap. Drug Carrier Syst., 7 No. 4, 275-308 (1991). These references were transmitted by the International Bureau.
Torchilin, V.p., et al. The antibody linked chelating polymers for nuclear therapy and diagnostics. Crit. rev. Therap. drug Carrire Syst. 1991., vol. 7, No. 4, pp. 275-308. Abstract Only.* *
Vuillez et al., "Two-step immunoscintigraphy for non-small cell lung cancer staging using a bispecific anti-CEA/anti-indium-DTPA antibody and an indium-111-labeled DTPA dimer", J. Nuc. Med., 38 No. 4, 507-511 (1997). These references were transmitted by the International Bureau.

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8329873B2 (en) 2002-04-05 2012-12-11 The Regents Of The University Of California Bispecific single chain Fv antibody molecules and methods of use thereof
US20060099205A1 (en) * 2002-04-05 2006-05-11 The Regents Of The University Of California Bispecific single chain FV antibody molecules and methods of use thereof
US7332580B2 (en) 2002-04-05 2008-02-19 The Regents Of The University Of California Bispecific single chain Fv antibody molecules and methods of use thereof
US8980258B2 (en) 2002-04-05 2015-03-17 The Regents Of The University Of California Bispecific single chain Fv antibody molecules and methods of use therof
US20090010840A1 (en) * 2002-04-05 2009-01-08 The Regents Of The University Of California BISPECIFIC SINGLE CHAIN Fv ANTIBODY MOLECULES AND METHODS OF USE THEREOF
US20040071696A1 (en) * 2002-04-05 2004-04-15 The Regents Of The University Of California Bispecific single chain Fv antibody molecules and methods of use thereof
US7332585B2 (en) 2002-04-05 2008-02-19 The Regents Of The California University Bispecific single chain Fv antibody molecules and methods of use thereof
US8003373B2 (en) 2003-04-25 2011-08-23 Medtronic, Inc. Optical detector for enzyme activation
US8940522B2 (en) 2003-04-25 2015-01-27 Medtronic, Inc. Optical detector for use in therapy
US20090252731A1 (en) * 2003-07-01 2009-10-08 Immunomedics, Inc. Multivalent Carriers of Bi-Specific Antibodies
US20110223645A1 (en) * 2003-07-01 2011-09-15 Immunomedics, Inc. Multivalent Carriers of Bi-Specific Antibodies
US8188239B2 (en) 2003-07-01 2012-05-29 Immunomedics, Inc. Multivalent carriers of bi-specific antibodies
US20050100543A1 (en) * 2003-07-01 2005-05-12 Immunomedics, Inc. Multivalent carriers of bi-specific antibodies
US7951921B2 (en) 2003-07-01 2011-05-31 Immunomedics, Inc. Multivalent carriers of bi-specific antibodies
US20100196265A1 (en) * 2006-11-21 2010-08-05 Adams Gregory P Anti-egfr family antibodies, bispecific anti-egfr family antibodies and methods of use thereof
US8580263B2 (en) 2006-11-21 2013-11-12 The Regents Of The University Of California Anti-EGFR family antibodies, bispecific anti-EGFR family antibodies and methods of use thereof

Also Published As

Publication number Publication date
WO1998038513A1 (en) 1998-09-03
EP0981748A4 (en) 2002-09-18
US20020119582A1 (en) 2002-08-29
EP0981748A1 (en) 2000-03-01
US20020031781A1 (en) 2002-03-14

Similar Documents

Publication Publication Date Title
US5914241A (en) Assays and kits for detecting analytes in the presence of cross-reacting substances
US6451980B1 (en) Signal enhancement of bispecific antibody-polymer probe for immunoassay use
US4731326A (en) Disease diagnosis by detection of shed normal tissue antigens
US20070166776A1 (en) Immunoassay and kit for an early and simultaneous detection of biochemical markers in a patient's sample
JPS6120867A (en) Sandwich test for antibody-lectin
EP1238284B1 (en) Diagnostic assay for stroke
WO2012175602A2 (en) Elisa for calprotectin
JP3055789B2 (en) Assay for alkaline phosphatase from bone
JP4334065B2 (en) Reduction of immunoassay interference by substances derived from the framework regions of antibodies.
US6255060B1 (en) Method of detecting protein by immuno RNA
US5646002A (en) Method for increasing the sensitivity of assays for target ligand
CA2405448A1 (en) Method of examining cancer by assaying autoantibody against mdm2 and reagent therefor
US6406858B1 (en) System for the reduction of interferences in immunoassays
JP2019510219A (en) Immunoassay controls and uses thereof
WO1997049994A1 (en) Method and kit for the diagnosis of troponin i
JPH11507014A (en) Immunoassay using antibodies directed to the reaction products of the enzymes used as labels
US5593898A (en) Diagnostic method for the immunological determination of NCAM
JP2000180447A (en) Method for deciding acute myocardial infarction and deciding reagent
JP3470936B2 (en) Immunoassay method and its reagent
EP0328664B1 (en) Method for assaying basic fetal protein in urine and assaying kit therefor
JP2001004632A (en) Detection method for carcinoembryonic antigens having varied sugar chain structure
JPH01224666A (en) Method of measuring immune complex
US5179000A (en) Method for assaying basic fetoprotein in urine and assay kit therefor
JP2520465B2 (en) Multi-labeled antibody
Haik et al. The Detection of the Severity of Acute Myocardial Infarction (AMI) Using Magnetic Microspheres

Legal Events

Date Code Title Description
AS Assignment

Owner name: KHAW, BAN-AN, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:NARULA, JAGAT;REEL/FRAME:010997/0001

Effective date: 20000526

AS Assignment

Owner name: BIOSPECIFIC LLC, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KHAW, DR. BAN-AN,;REEL/FRAME:012035/0865

Effective date: 20010515

STCF Information on status: patent grant

Free format text: PATENTED CASE

AS Assignment

Owner name: KHAW, DR. BAN-AN, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BIOSPECIFIC LLC;REEL/FRAME:015226/0322

Effective date: 20040405

Owner name: KHAW, DR. BAN-AN, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BIOSPECIFIC LLC;REEL/FRAME:015226/0374

Effective date: 20040405

REMI Maintenance fee reminder mailed
FPAY Fee payment

Year of fee payment: 4

SULP Surcharge for late payment
AS Assignment

Owner name: AKRIVIS TECHNOLOGIES, LLC, MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KHAW, BAN-AN;REEL/FRAME:023263/0174

Effective date: 20090312

FPAY Fee payment

Year of fee payment: 8

FPAY Fee payment

Year of fee payment: 12